Methods of preparing myxomavirus compositions

ABSTRACT

The present invention relates to monoparamunity inducers based on paramunizing viruses or viral components of a myxomavirus strain from rabbits with typically generalizing disease, to a method for the production thereof and to the use thereof as medicaments for the regulatory optimization of the paramunizing activities for the prophylaxis and therapy of various dysfunctions in humans and animals.

This application is a continuation of application Ser. No. 12/039,120,filed Feb. 28, 2008 now U.S. Pat. No. 7,939,085, which is a continuationof U.S. application Ser. No. 10/587,082, filed on Jul. 21, 2006, nowU.S. Pat. No. 7,494,799, which was the National Stage of InternationalApplication PCT/EP2005/000582, filed Jan. 21, 2005, which claims thebenefit of German Application 10 2004 003 572.5, filed Jan. 23, 2004.All of these applications are incorporated herein by reference.

The invention relates to monoparamunity inducers based on paramunizingviruses or viral components, characterized in that the viruses or viralcomponents are derived from an attenuated rabbit myxomavirus strain, toa method for producing the monoparamunity inducers and to the usethereof as medicaments.

The endogenous immune system of highly developed organisms, especiallythat of mammals and birds, includes an antigen-specific and anantigen-nonspecific part. Both parts of the immune system are linkedtogether and moreover interact with one another. The antigen-specificmechanisms are responsible for building up immunity, and theantigen-nonspecific are responsible for building up paramunity.Paramunity refers to the state of a well regulated and optimallyfunctioning nonspecific defense system linked to a rapidly developing,time-limited, increased protection from a large number of differentpathogens, antigens and other noxae. The basis for the development ofparamunity are, for historical and functional reasons, the nonselectiveand conditionally selective paraspecific defense mechanisms which areold from the phylogenetic viewpoint and are called primitive.

The paraspecific activities of the antigen-nonspecific immune system(also: “innate immune system”) include nonselective protective elementssuch as, for example, foreign material-consuming organelles, andconditionally selective protective elements such as, for example, micro-and macrophages, natural killer cells, dendritic cells and solublefactors such as cytokines, which show pathogen-nonspecific orantigen-nonspecific reactions according to their origin.

Paraspecific activities are to be observed in the relevant organismimmediately after antigen contact, whereas the effects of theantigen-specific immune system appear only after days or weeks.

In the more highly organized life forms, time is additionally gainedthereby in order to build up specific defense systems against the noxaewhich it has not yet been possible to eliminate and have antigenicproperties.

The benefits of paramunization i.e. the paraspecific activities of theimmune system for prophylaxis and therapy in a patient have becomeincreasingly clear since its development (Anton Mayr, “Paramunisierung:Empirie oder Wissenschaft”, Biol. Med. edition 26(6): 256-261, 1997).The paraspecific defenses make it possible for the organism to defenditself immediately on confrontation with a wide variety of foreignmaterials, infectious pathogens, toxins and transformed endogenouscells.

There are close interactions between the paraspecific and the specificactivities of the immune system, with the flow of information usuallyproceeding from the initially reacting paraspecific part to the specificpart, with a later onset, of the immune system (e.g. with antigenmediation). In the event of infections with particularly virulentpathogens, the paraspecific defenses of the organism are able in thisway to cover the time until specific immunity develops (e.g. antibodies,immune cells).

The paraspecific immune defenses are a physiological process and can bedefined as “primary control” in the confrontation with the environment.They are indispensable not only for lower organisms but in particularalso for the more highly developed and highly developed vertebrates.Primary congenital defects in this biological defense system lead tolife-threatening situations. An example which may be mentioned is the“Chediak-Steinbrinck-Higashi syndrome” in humans, which is characterizedby granulocyte deficits and dysfunctions of natural killer cells (NKcells) and in most cases leads to the death of the patient by completionof the 10th year of life.

The condition of paramunity is characterized by an increased rate ofphagocytosis, an increased function of the spontaneous cell-mediatedcytotoxicity (NK cells) and increased activity of other lymphoreticularcells. At the same time there is release of particular cytokines whichhave stimulating and/or inhibiting effects (e.g. via repressormechanisms) both with the cellular elements and with one another. Thisclosely linked and stepwise responding biological system of paramunitywith its various acceptor, effector and target cells and thesignal-transmitting cytokines is moreover thoroughly connected to thehormonal and nervous systems. It thus represents an importantconstituent of the communication, interaction and regulation network.

Paramunity is induced by paramunization. By this is meant thepharmacological activation of the cellular elements of the paraspecificpart of the immune system and the production, associated therewith, ofcytokines, with the aim of eliminating dysfunctions, rapidly increasingthe non-pathogen- and non-antigen-specific protection of an individual(optimal bioregulation), eliminating an immunosuppression orimmunodeficiency which has arisen from the consequences of stress orotherwise (e.g. pharmacologically), repairing deficits and/or acting asregulator between the immune, hormonal and nervous systems. This meansthat certain nonspecific endogenous defense processes can be increased,supplemented or else depressed, depending on the type of paramunizationand the responsiveness, such as, for example, the health status of thepatient.

Paramunity inducers are used for the paramunization and must meetcertain criteria of harmlessness and efficacy, thus differing fromimmunostimulants. The paramunity inducer per se is not comparable eitherto an antibody or to a chemical, an antibiotic, vitamin or hormone. Onthe contrary, it activates by a stepwise mechanism the paraspecificimmune system, so that the latter sufficiently mobilizes cellular andhumoral defense mechanisms. The paramunity inducer in this case has bothregulating and repairing effects on the immune defenses. Concerning themode of action of paramunity inducers, it is known that they are takenup by phagocytic cells (acceptor cells) which are thus activated andrelease mediators which in turn mobilize effector cells. The latterfinally switch on the regulatory mechanisms of the paraspecificdefenses.

Multiple paramunity inducers based on combinations of two more poxviruscomponents derived from different poxvirus strains with paramunizingproperties are described in European patent EP 0 669 133 B1.

The present invention is based on the paramunizing properties ofattenuated myxomaviruses and/or their viral constituents.

Attenuation refers to the modification of the properties of aninfectious pathogen which lead to weakening of the pathogen(“attenuate”=weaken, mitigate). Alterations in infectious pathogensfrequently occur spontaneously in nature, it being possible for thetimespans to extend over many centuries.

The ability of infectious pathogens to change in order to adapt toenvironmental changes can be utilized experimentally, and the timespannecessary for attenuation can be drastically shortened for example bylong-term passages in certain host systems. Attenuation has beenutilized to date to obtain avirulent inoculation strains and harmlessparamunity inducers.

Attenuation normally leads to loss of virulence and contagiousness,reduction in the immunizing properties and the host range, and to smallchanges in the pathogen genome with the occurrence of deletions,preferably in the terminal regions. There is usually parallel increasein the paramunizing activities of the modified pathogen.

In rare cases, an attenuation may, especially when an experimentalattenuation by genetic manipulations is attempted, also lead to anincrease in the virulence and contagiousness.

Myxomaviruses are the pathogens of myxomatosis, a contagious systemicviral disease of wild and domestic rabbits which progresses in cyclesand is characterized by generalized, in some cases hemorrhagicsubcutaneous edemas on the head and over the entire body, withpreference for the anal region, the vulva and the tube, unlike any otherinfectious disease. Introduction of myxomatosis into a countrypreviously free of the disease results in rapid and fatal progression.After the virus has become endemic, the character of the disease changesuntil the infections are clinically inapparent (Mayr A.: MedizinischeMikrobiologie, Infections—and Seuchenlehre, 7th edition, Enke-Verlag,Stuttgart, 2002).

The disease is widespread among American cottontail rabbits of the genusSylvilagus which occupy exclusively the New World. These wild rabbitsform the only natural reservoir of the disease. The infection takes amild form in them. By contrast, the disease has an almost 100% mortalityin European wild and domestic rabbits of the genus Oryctolagus, whichare also naturalized in Australia, when the pathogen is introduced.

The natural host range of the myxomavirus (genus Leporipoxvirus) hasnarrow limits. In general, the virus replicates only in Americancottontail rabbits and in European domestic and wild rabbits. However, afew infections in European wild hares have also been observed. Attemptsat transmission to other species and to humans had negative results.

The present invention is based on the object of providing novelmonoparamunity inducers for human medicine and veterinary medicine. Afurther object of the present invention is to provide a method forproducing such monoparamunity inducers. It is additionally an object ofthe present invention to provide pharmaceutical compositions for use asmedicaments based on monoparamunity inducers.

Accordingly, the present invention relates to monoparamunity inducersbased on paramunizing viruses or viral components, characterized in thatthe viruses or viral components are derived from an attenuated rabbitmyxomavirus strain. The viral components preferably include paramunizingviral envelopes or aberrant forms of viral envelopes of an attenuatedmyxomavirus strain. Preferred strains having the paramunizing propertiesof the invention are the strains M-2, M-7, Lausanne,Aust/Uriarra/Verg-86/1. The strains M-7, Lausanne,Aust/Uriarra/Verg-86/1 are also suitable for producing live vaccinesbecause they have undergone only partial attenuation of their virulencein order to have an adequate immunizing effect.

A monoparamunity inducer based on the myxomavirus strain M-2 isparticularly preferred. An attenuated myxomavirus strain produced by themethod of the invention described hereinafter has been deposited at thedepository of the Public Health Laboratory Service (PHLS), Centre forApplied Microbiology & Research, European Collection of Animals CellCultures (ECACC), Porton Down, Salisbury, Wiltshire, SP4 0JG, UnitedKingdom with the deposit number 03121801.

The invention further relates to a method for producing monoparamunityinducers based on an attenuated rabbit myxomavirus strain. For thispurpose, initially myxomaviruses are isolated from infected tissue of arabbit typically suffering from a generalized myxomatosis. The virus issubsequently adapted to a permissive cell system, i.e. to a cellmaterial which permits replication of the virus, such as, for example,cell cultures, incubated chicken eggs or else experimental animals. Itis possible in particular to use cells of the natural host or of aspecies closely related to the host for the adaptation. Examples ofsuitable permissive cell systems for myxomaviruses are chick embryofibroblasts (CEF) as well as cell cultures produced from rabbit kidneysor testes.

It is preferred according to the invention to adapt the isolatedmyxomavirus to the chorioallantoic membrane (CAM) of incubated chickeneggs over one or more passages, preferably over 2 to 6 passages, andparticularly preferably over 3 passages. For this adaptation, theisolated viruses are inoculated onto the CAM and replicated by passagingon the CAM.

The myxomaviruses are preferably initially isolated from infected tissueby replication in a permissive cell system. For this purpose it ispossible to inoculate a permissive cell system for example with infectedtissue homogenate obtained by disruption. Subsequently, the virusesobtained by the initial replication are either adapted further to thesame permissive cell system, which was already used for the isolation,or another, further permissive cell system is used. Adaptation of thevirus to the same permissive cell system type which is also used for theisolation is preferred. Thus, preferably myxomaviruses from infectedtissue are isolated by replication in a permissive cell system andsubsequently adapted to the permissive cell system by further passages.Initial culturing and isolation of the myxomaviruses by replication onthe CAM of incubated chicken eggs and subsequent adaptation of the viruson the CAM over further passages, preferably over a further 2 passages,is particularly preferred. However, it is also possible alternatively touse the allanotoic fluid of incubated chicken eggs for the culturingand/or adaptation.

The actual attenuation then takes place by long-term passages on one ormore permissive cell cultures, until an attenuation or the desireddegree of attenuation of the virus is reached. It is possible for thispurpose firstly to test various permissive cell systems for replicationof the virus and subsequently to select one or more cell systems inwhich the highest infectious titers are reached for further passages.Both primary and secondary cell cultures as well as permanent andcontinuous cell lines are suitable for attenuation by long-termpassages. Thus, attenuation can take place by replication in primary orsecondary chick embryo fibroblast (CEF) cultures or in cultures ofpermanent CEF cells.

Preferred according to the invention for attenuation of the myxomavirusis to passage or replicate the virus in a permanent cell culture, inparticular a Vero cell culture, preferably over 80 to 150 passages andparticularly preferably over 120 passages. Alternatively oradditionally, the virus is passaged according to the invention on abinary permanent cell line, in which case AVIVER cells are preferablyused. These cells or cell culture has been obtained by cell fusionbetween chick embryo fibroblasts (CEF) and Vero monkey kidney cells. Forthe myxomavirus attenuation of the invention, the isolated and adaptedviruses are preferably passaged in a first step in Vero cell cultures,the viruses are then transferred into a binary AVIVER cell culture andreplicated therein preferably over 10 to 50 passages, in particular over20 to 30 passages, and particularly preferably over 25 passages.

The attenuated myxomavirus can then be additionally replicated overfurther attenuation passages. Further replication of the virusespreferably takes place over further passages in Vero monkey kidneycells, in particular over 100 to 200 passages.

A particularly preferred further method step is additional inactivationof the attenuated myxomavirus. Inactivation can take place by chemicaltreatment, irradiation, the action of heat or pH, in particular by achemical treatment with beta-propiolactone. Treatment of the attenuatedmyxomaviruses with beta-propiolactone increases the paraspecificactivities further, while the immunizing properties which are stillpresent where appropriate after the attenuation are lost.

A particularly preferred embodiment of the method of the inventionincludes the following steps:

-   -   isolation of myxomaviruses from the infected tissue of a rabbit        suffering typically from generalized myxomatosis by replication        on the chorioallantoic membrane (CAM) of incubated chicken eggs        and subsequent adaptation of the virus to the CAM over a further        2 passages;    -   attenuation of the isolated viruses by passaging in Vero cell        cultures, preferably over 120 passages;    -   transfer of the viruses into a binary AVIVER cell culture, where        the AVIVER cells have been obtained by cell fusion between chick        embryo fibroblasts (CEF) and Vero monkey kidney cells, and        attenuation of the virus in this cell culture over 10 to 50,        preferably 25, passages,    -   subsequent transfer of the virus back onto Vero monkey kidney        cells and replication of the viruses by further attenuation        passages in the Vero cells, preferably over about 150 passages;        and optionally    -   inactivation of the attenuated myxomaviruses by treatment with        beta-propiolactone, this treatment increasing the paraspecific        activities, while the immunizing properties still present after        the attenuation are lost.

Attenuation by long-term passaging is usually concluded by 3 to 5 plaqueend dilutions. After various clones have been obtained and tested, theclones selected for further replication are those with which the highestinfectious titers are achieved and with which a high paramunizingactivity—e.g. in the VSV challenge test in the baby mouse—are detected.This procedure is intended to ensure in particular that geneticallyuniform virus material is provided for further use.

The term “attenuation” as used in connection with this invention refersto the experimental modification of the originally virulent myxomavirusinto the modified form, with a simultaneous increase in the paramunizingproperties. The attenuation is detectable through one or more of thefollowing properties:

-   -   reduction or weakening or loss of virulence for European        domestic and wild rabbits (genus Oryctolagus caniculus csp.),    -   weakening or loss of contagiousness,    -   restriction of the host range in cell cultures,    -   alteration of the immunizing properties    -   acquisition of paramunizing, short-term protective activities.

An attenuation may lead to deletions in the terminal region of themyxomavirus genome. An increasing degree of attenuation is frequentlyobserved to be associated with an increasing number of deletions in theviral genome.

The degree of attenuation can be checked and monitored during thepassages by appropriate suitable activity tests as known in the art(cf., for example, U.S. Pat. No. 6,805,870, column 12, and the furtherreferences cited therein) and by cloning.

The present invention further relates to attenuated myxomavirusesobtainable by the method of the invention, pharmaceutical compositionsincluding the attenuated myxomavirus or the myxomavirus mono-paramunityinducer of the invention, and to the use of the myxomavirusmonoparamunity inducers for activating the paraspecific immune system ina mammal and to the use of the attenuated virus for producing acorresponding medicament.

Because of the surprising paramunizing properties, the myxomavirusmonoparamunity inducers of the invention are suitable for the treatmentand/or for the prophylaxis of immune system dysfunctions,immuno-suppression, immunodeficiency disorders, dysfunctions ofhomeostasis between the hormonal, circulatory, metabolic and nervoussystems, threatened neonatal infection, neoplastic diseases, viraldiseases, bacterial diseases, therapy-resistant infectious factordiseases, viral and bacterial mixed infections, chronic manifestationsof infectious processes, liver disorders of various etiologies, chronicskin disorders, herpetic diseases, chronic hepatitides, influenzalinfections, endotoxin damage.

The monoparamunity inducers of the invention are generally harmless tothe environment and effective in the sense of paramunization for mammalssuch as, for example, humans, horses, dogs, cats, pigs, for birds andalso for reptiles such as, for example, lizards, snakes, chelonians.They are therefore particularly suitable for use in human and veterinarymedicine.

In addition, the monoparamunity inducers of the invention display a verygood paramunizing activity with high potency. They can be produced bythe method of the invention in a suitable manner and are safe for use inthe medical sector. The attenuation of the myxomaviruses reduces theimmunizing properties of the myxomaviruses while the paraspecificactivities increase. The monoparamunity inducers of the inventiontherefore have paramunizing, but no immunizing, properties, makingmultiple and continuous use possible. These paramunizing properties ofthe rabbit myxomavirus and of its paramunizing viral components aresurprising and were not predictable.

The term “paramunization” as used in connection with this inventionrefers to the pharmacological activation of the cellular elements of theparaspecific immune system and the production or release, associatedtherewith, of cytokines with the aim of eliminating dysfunctions,rapidly increasing the non-pathogen- and non-antigen-specific protectionof an individual, and having a regulatory effect between the immune,hormonal, nervous and vascular systems. Paramunization leads to theprotected state of paramunity.

The term “paramunity” as used in connection with this invention refersto the actively acquired state of an optimally regulated and functioningparaspecific defense system, associated with a rapidly developing,time-limited protection from a large number of pathogens, antigens andother noxae. The phagocytosis rate, the function of the NK cells(natural killer cells) and the activity of other lymphoreticular cells(e.g. dendritic cells) are raised to the physiological optimum.

The term “paramunity inducer” as used in connection with this inventionrefers to a pyrogen-free, nontoxic medicament which is intended to beused in humans and animals to generate and regulate endogenous defenseand protective mechanisms in the sense of paramunization.

The term “myxomavirus monoparamunity inducer” as used in connection withthis invention refers to a medicament which is based on attenuatedrabbit myxomaviruses or an attenuated myxomavirus strain, including theparamunizing viral components and the constituents thereof which producethe state of paramunity in an organism, preferably in a mammal (e.g.human).

The term “myxomavirus” as used in connection with this invention refersto the species of the myxomatosis virus of the genus Leporipoxvirus. Themyxomavirus belongs to the subfamily of Chordopoxviridiae and to thefamily of Poxviridae (poxviruses).

The term “paramunizing viral components” as used in connection with thisinvention includes a large number of viral structures derived from amyxomavirus having paramunizing properties, for example viable orinactivated freshly isolated myxomaviruses, viable or inactivatedrecombinant myxomaviruses derived from freshly isolated myxomaviruses,viral envelopes, the removed envelopes and cleavage products andaberrant forms of these envelopes, individual native or recombinantpolypeptides or proteins, in particular membrane and surface receptorswhich occur in freshly isolated myxomaviruses or are recombinantlyexpressed by a genetically modified myxomavirus or a part of its geneticinformation.

Tables 1 to 4 summarize the clinical results with the myxomavirusmonoparamunity inducer PIND-MYXO based on the attenuated myxomatosiscell culture virus, strain M-2, in humans.

Table 1 shows the clinical results on prophylactic use of themyxomavirus monoparamunity inducer PIND-MYXO in humans.

Table 2 shows the clinical results on therapeutic use of the myxomavirusmonoparamunity inducer PIND-MYXO in humans.

Table 3 shows the effect of paramunization with myxomavirusmonoparamunity inducer (PIND-MYXO) in patients with low immuneparameters (7 days after PIND-MYXO administration).

Table 4 shows the effect of paramunization with myxomavirusmonoparamunity inducer (PIND-MYXO) in patients with elevated immuneparameters (7 days after PIND-MYXO administration).

The invention is based on the surprising finding that attenuated rabbitmyxomaviruses or their paramunizing constituents are able to induce verygood paramunizing properties in a recipient organism which lead to theprotected state of paramunity. The basis for this invention was thefirst successful attenuation of rabbit myxomaviruses in cell cultures.

The monoparamunity inducers of the invention are preferably based onlyophilized, attenuated and inactivated rabbit myxomaviruses or theirparamunizing viral components. The attenuated myxomaviruses or theirviral components of the invention are preferably derived from onemyxomavirus strain or a plurality of different attenuated myxomavirusstrains. It is preferred in this connection for the monoparamunityinducers of the invention to include combinations of one or moremyxomavirus strains or their paramunizing viral components.

The paramunizing properties induced in a mammal such as, for example, inhumans through administration of myxomavirus monoparamunity inducer areparticularly beneficial for eliminating dysfunctions, for increasing thenon-antigen-specific protection of an individual, for eliminating animmunosuppression or immuno-deficiency which has arisen from theconsequences of stress or in other ways (e.g. pharmacological) and inorder to have regulatory effects between the immune, hormonal andvascular systems.

The invention is further based on successful attenuation of amyxomavirus strain by passaging through cell cultures, with the virulentand/or immunizing properties of the myxomavirus strain being reduced orlost. Additional inactivation of the myxomaviruses can moreover takeplace by irradiation, the action of heat or pH, or, particularlypreferably, by a chemical treatment with beta-propiolactone. Themonoparamunity inducers are based on attenuated, lyophilizedmyxomaviruses, with individual viral components of a myxomavirus whichare suitable for inducing paramunizing activities in an organism alsobeing encompassed by the invention.

It is intended below to describe one embodiment of the production methodof the invention for monoparamunity inducers based on an isolated andattenuated rabbit myxomavirus strain via cell culture passaging. Theproduction method is moreover not restricted to this preferred strain,but can be applied in the same way to other rabbit myxomavirus strains.Also encompassed by the present invention are recombinant forms of amyxomavirus strain which have been produced by genetic modification.Preference is given in this connection to recombinant myxomavirusstrains in which one or more segments in the genome which code forcytokine receptors has been modified by a modification in the form of anaddition, substitution or deletion, with the receptor properties of thecytokine receptor being lost through the modification. These arepreferably the gene segments which code for the receptors forinterferons (IFN), interleukins (IL) and tumor necrosis factors (TFN),in particular for IFN-α-R, IFNγ-R, TNF-R, IL-1-R, IL-2-R, IL-6-R andIL-12-R.

In addition, the numerical values stated herein concerning theincubation time or the number of passages over cell cultures are notintended to be regarded as restrictive. Slight modifications of theseparameters and modifications evident to the skilled worker and alsoleading to a preparation of attenuated myxomaviruses are equallyencompassed by this invention.

A preferred embodiment of the present invention relates to thesuccessful attenuation of the myxomavirus strain M-2. The myxomavirusstrain M-2 was isolated from European wild rabbits suffering frommyxomatosis (Herrlich A., Mayr A. and Munz E.: “Die Pocken”, 2ndedition, Georg Thieme Verlag, Stuttgart, 1967). The altered skin cellsobtained from the subcutaneous tissue of the diseased rabbit are, afterdisruption, inoculated onto the chorioallantoic membrane (CAM) ofchicken eggs which have been incubated preferably for 10-12 days. Themyxomaviruses are further replicated and adapted over 2 to 6 passages,preferably over 3 passages, on the chorioallantoic membrane (CAMpassages; for method, see Herrlich A., Mayr A. and Munz E.: “DiePocken”, 2nd edition, Georg Thieme Verlag, Stuttgart, 1967). The 2nd to6th passage, preferably the third CAM passage, serves as startingmaterial for the further attenuation of the myxomavirus in cellcultures. The attenuation takes place after adaptation of the viruses inthe chorioallantoic membrane (evident from typical foci on thechorioallantoic membrane) in 3 stages. In stage 1, 80 to 150, preferably120, continuous so-called end-dilution passages take place in Vero cells(Vero cells, ATCC CCL-81). The virulence of the myxomavirus which hasundergone these passages is weakened.

In a 2nd stage after the 80th to 150th passage, preferably after the120th passage, in Vero cells, the viral suspension is transferred toso-called AVIVER cells and continued over 10 to 50 passages, preferablyover 20 to 30, in particular over 25, passages. AVIVER cells areobtained by cell fusion between chick embryo fibroblasts (CEF) and Verocells and are referred to as binary permanent cell culture.

The last passage over AVIVER cells, preferably the 25th passage, istransferred back to Vero cells and is continued in the 3rd stage ofattenuation for a further 100 to 200 passages, preferably about 157passages, in Vero cells. In this way, the myxomavirus is replicated overa total of more than 300 cell culture passages. After the 3rd stage ofreplication of the myxomavirus in cell cultures, the myxomavirus issufficiently attenuated.

The Vero cell cultures and the AVIVER cells are preferably culturedusing a completely synthetic medium, particularly preferably the MEMmedium (minimal essential medium) plus 5 to 20%, preferably 10%, BMS(serum substitute medium) and 5 to 20, preferably 10%, lactalalminhydrolysate. The virus medium used after exchange with the culturemedium is preferably MEM medium with 5 to 20%, preferably with 10%,lactalalbumin hydrolysate, without BMS or without fetal calf serum andwithout antibiotics. All the production methods are preferably carriedout at pH values of 7.0 to 8.0, preferably at a pH of 7.25. Virusharvests with titer from 10^(5.0) to 10^(7.5), preferably of at least10^(6.5) TCID₅₀/ml (TCID₅₀=50% tissue culture infectious dose) arepreferably suitable as starting material for producing themonoparamunity inducer PIND-MYXO of the invention.

Replication of the myxomavirus in Vero cells leads to a typicalcytopathic effect (cpE) which is ultimately characterized by adestruction of the infected cells (lysis). Inoculation with a dose ofabout 10 MOI (multiplicity of infection) results after a short roundingphase (1-2 days) in reticulated cell structures for about 3 days and inlysis of the cells after about 5 days. The 301st passage in Vero cellshad an infectious titer of about 10^(6.5) TCID₅₀/ml.

The attenuated myxomavirus is inactivated by a chemical treatment withbeta-propiolactone at a concentration of 0.01-1% beta-propiolactone,preferably at a concentration of 0.05% beta-propiolactone. Thisinactivation leads to a complete loss of the immunizing properties whichare still present where appropriate, while the paraspecific activitiesare not only retained but in fact significantly increase.

For further processing of the attenuated and inactivated myxomavirusesto a myxomavirus monoparamunity inducer (PIND-MYXO), the virus startingmaterial used for the virus inactivation should have a viral titer ofabout 10^(5.0) to 10^(7.0), preferably of at least 10^(6.5), TCID₅₀/ml.

Purification preferably takes place by centrifugation at low revolutions(e.g. 1000 rpm). After the centrifugation, 0.5-10% succinylated gelatin(e.g. polygeline, e.g. from Hausmann, St Gallen/Switzerland), preferably5% succinylated gelatin, is added. The resulting mixture cansubsequently be lyophilized in 1.5 ml portions in appropriate sterileglass vials or ampoules and, if required, dissolved in distilled water.A volume of 0.5-2 ml, preferably of 1.0 ml of the lyophilisate dissolvedin distilled water corresponds to an inoculation dose for humans onintramuscular administration (see also Mayr A. and Mayr B.: “Von derEmpirie zur Wissenschaft”, Tierärztl. Umschau, edition 56: 583-587,2002).

The attenuation can be detected clinically through the loss of virulencefor European domestic and wild rabbits (genus Oryctolagus caniculuscsp.), through a loss of contagiousness, through a virtually completerestriction of the host range in cell cultures and through thealteration in the immunizing properties.

The lyophilized product can be stored at temperatures of, preferably,about +4° C. or at lower temperatures, preferably about −60° C., withstability for an unlimited time.

It was demonstrated by gene technology investigations of the preparedattenuated myxomaviruses that multiple deletions had occurred in themyxomavirus genome. In the case of the initial strain M-2, themyxomavirus genome consists of a single linear deoxyribonucleic acid(DNA) with a total length of about 160 kilobases (kb), which codes forseveral hundred proteins (Herrlich A., Mayr A. and Munz E.: “DiePocken”, 2nd edition, Georg Thieme Verlag, Stuttgart, 1967). Thesequences of the terminally located inverted repeats (terminal invertedrepeats, TIR) are situated at about 11 kb of the genome segment(McFadden, G. and Graham K.: “Modulation of cytokine networks by poxvirus”, Virology, edition 5: 421-429, 1994).

Thus, it has been found that the attenuation of the myxomaviruses overcontinuous Vero cell passages has led to a loss of the coding genesegments for the receptors for interferon α and γ (IFN α, IFNγ), fortumor necrosis factor (TNF) and for the interleukins (IL) 1, 2, 6 and12. It is of interest that these cytokines belong to the paraspecificdefense factors of the nonspecific immune system. The cytokines areneutralized by binding to the corresponding viral receptors, so that thevirus is able to replicate unimpeded. The deletions of gene segmentswhich code for the abovementioned cytokine receptors relate mainly tothe terminal regions of the DNA. However, it was additionally possibleto detect deletions which occurred during the AVIVER cell passages inthe conserved part of the DNA. These deletions relate to two genes whichcode for an immune epitope and virulence gene. Such geneticmodifications are presumably one of the reasons for the decrease in theimmunizing, i.e. antigen-specific, activities and the simultaneousincrease in the paraspecific activities of the attenuated myxomavirus.

The immunizing epitopes and the paraspecific and nonspecific epitopesare in competition. A decrease in the first-mentioned peptides orproteins therefore leads to an increase in the effect of theparaspecific activities. Residues of immunizing and virulence-increasingproteins are eliminated in the preparation of monoparamunity inducers bythe method described above for inactivating the attenuatedmyxomaviruses.

The monoparamunity inducer of the invention, also called PIND-MYXO, isbased on the use of attenuated myxomaviruses or the paramunizingconstituents thereof and is suitable on the basis of its paramunizingproperties for the following prophylactic or therapeutic indications ina patient:

-   -   infectious factor diseases and mixed infections, chronic        manifestations of infectious processes, refractory recurrent        infections and chemotherapy-resistant bacterial and viral        infections    -   weakened defenses and dysregulations in the defense system of an        organism    -   threatened neonatal infection    -   adjuvant therapy for certain neoplastic diseases, e.g.        prevention of metastasis, reduction of side effects due to        chemo- and radiotherapy    -   regulation of homeostasis between the hormonal, circulatory,        metabolic and nervous systems.

The paramunity inducers of the invention can be administeredparenterally or locally to mammals, including humans, birds andreptiles. Local administration of paramunity inducers specificallystimulates the paraspecific defense mechanisms in the mucous membranesand in the skin. However, there is also a certain systemic effect. Bycontrast, parenterally applied paramunizations scarcely influence thelocal defense mechanisms in the skin and mucous membrane, preferablyhaving a systemic effect.

Side effects do not occur even with numerous parenteral administrationscarried out continuously in humans and animals. The indications for theuse of PIND-MYXO are the same for animals and for humans. At the sametime, in problem operations, specifically in the management of horses,pigs, dogs and cats, paramunization of neonates immediately on the dayof birth and preferably on the first and possibly also the second dayafter birth is advisable. The single dose is about 0.5 to 5 ml of thedissolved lyophilisate, in horses and pigs the single dose is preferably2 ml and in dogs and cats is preferably 0.5 ml on parenteraladministration. It is advisable according to the invention to administerPIND-MYXO parenterally one day before and/or at the same time asspecific protective inoculations in order to avoid secondary reactionsand to assist the immunization on administration of vaccines.

One embodiment of the invention relates to the production of apharmaceutical composition for local administration to induce paramunityin the skin and mucous membranes. The pharmaceutical compositionpreferably relates to a buccal or suckable tablet based on constituentsof an attenuated and inactivated myxoma cell culture virus. The buccaltablets of the invention are preferably produced with addition ofsorbitol, polyethylene glycol 6.00, potassium hydrogenphosphate,Tyrospirol tablet essence, Kollidon 25 and magnesium stearate. PIND-MYXOcan, however, also be administered nasally, rectally or vaginally withsuitable carriers.

The following examples are preferred embodiments of the invention andserve to explain the subject matter of the invention further.

EXAMPLE 1

The myxomavirus from the edematous subcutis of a European wild rabbit(genus Oryctolagus) suffering in a typical manner from myxomatosis wasisolated as starting material for producing the monoparamunity inducerPIND-MYXO of the invention by culturing on the chorioallantoic membrane(CAM) of chicken eggs (Valo eggs) incubated for 10 days and was adaptedthree times by the method of Herrlich et al. in passages on the CAM(Herrlich A., Mayr A. and Munz E.: “Die Pocken”, 2nd edition, GeorgThieme Verlag, Stuttgart, 1967). The third CAM passage was adapted in a1st stage on Vero cells over 120 passages (ATCC CCL-81, WHO, AmericanType Culture Collection), replicated in a 2nd stage by intermediatepassages in AVIVER cell cultures, and cultured further in the 3rd phasein Vero cells. In total, about 300 passages aimed at attenuation werecarried out. After these continuous end-dilution passages, theoriginally virulent myxomavirus was attenuated.

The attenuated myxomavirus is replicated in Vero cells. The Vero cellcultures are cultured using a completely synthetic medium consisting ofMEM (minimal essential medium) plus 10% BMS (serum substitute medium)and 10% lactalbumin hydrolysate. The virus medium used after exchangewith the culture medium is only MEM with 10% lactalbumin hydrolysatewithout BMS or without fetal calf serum and without antibiotics. Allproduction methods are carried out at pH values above 7.25. Viralharvests with titers above 10^(6.5) TCID₅₀/ml serve as starting materialfor producing the monoparamunity inducer PIND-MYXO of the invention.Inactivation of the viral harvests with 0.05% beta-propiolactone andlow-speed centrifugation are followed by addition of 5% of succinylatedgelatin (polygeline) to the virus material before lyophilization.

The lyophilized product is stable at room temperatures and attemperatures of about 4° C. to −80° C. and can be kept without timerestriction preferably at about 4° C. or else about −60° C. A volume of1 ml of the lyophilisate dissolved in sterile distilled watercorresponds to an inoculation dose. Deep intramuscular or localadministration takes place (see examples 3, 4 and 5).

EXAMPLE 2

The PIND-MYXO inducer of the invention is administered in an analogousmanner to the description in example 1 in dry form (lyophilisate notdissolved) locally onto the mucous membranes of the upper respiratorytract, preferably nasally, three times a day for prophylaxis or therapy(1 ml per application) of multifactorial infections (e.g. influenzalinfections).

EXAMPLE 3

The PIND-MYXO inducer in liquid form produced as in example 1 is in ananalogous manner rubbed in cutaneously to improve the perfusion of theskin, to speed up the healing of wounds and to treat varicose veins andchronic venous insufficiency (leg ulcer) in humans. The lyophilisate canfor this purpose be taken up for example in greasy cream (e.g.Bepanthen, Linola fat), in which case the pH should be slightlyalkaline. This preparation should be prepared fresh for each use.Administration is carried out several times a day by manual rubbing intothe undamaged skin. Open wounds can be treated by dropwise applicationof the freshly dissolved product onto the wound regions. The treatmentshould take place each day until healed.

EXAMPLE 4

The monoparamunity inducer PIND-MYXO produced as in example 1 isanalogously administered parenterally to prevent secondary reactions andto improve the result of inoculation one day before and simultaneouslywith a protective inoculation with conventional specific vaccines.

EXAMPLE 5

The monoparamunity inducer PIND-MYXO produced as in example 1 isprocessed analogously to buccal or suckable tablets. The production anduse of the suckable tablets for local paramunization of the mucousmembranes of the ear, nose, throat and mouth is novel and a constituentof the invention. Via the activated mucous membranes of the mouth thereis not only a homing effect (migration of defense cells into mucousmembranes of other organ systems), but also a partial parenteralparamunization. The following production method has proved suitable forthe production of buccal and suckable tablets:

For the lyophilization, 5% Kollidon 25 (polyvinyl-pyrrolidone) is addedinstead of gelatin to the liquid inducer material. Urea, sorbitol,polyethylene glycol 6000 and magnesium stearate are required to producethe finished tablet. A recommended formula for a tablet with a weight of500.5 mg in weight is:

PIND-MYXO lyophilisate 65 mg Urea 50 mg Sorbitol 267 mg Polyethyleneglycol 6000 118 mg Magnesium stearate 0.5 mg Tablet weight 500.5 mg

The patient should take 4-6 tablets at regular intervals each day toachieve an optimal paramunization.

The following formula of a pharmaceutical composition has provedsuitable for producing buccal tablets:

PIND-MYXO lyophilisate 155 mg Sorbitol 360 mg Polyethylene glycol 6000300 mg Potassium dihydrogenphosphate (KH₂PO₄), 2 mg anhydrous Disodiumhydrogenphosphate (Na₂HPO₄), anhydrous 8 mg Tyrospirol essence tablets0.8 mg Magnesium stearate 20 mg Tablet weight 805.8 mg

The tablets slowly dissolve in the patient's mouth and can be swallowedafter dissolving.

The clinical test results with the monoparamunity inducer of theinvention, based on the lyophilisate of the myxomavirus strain M-2,which are compiled in tables 1 to 4 demonstrate the very goodparamunizing activities of myxomavirus lyophilisates in humans. Thesedata are equally applicable to other mammals as well as birds andreptiles.

TABLE 1 Clinical results with a monoparamunity inducer from attenuatedmyxoma cell culture virus in humans -prophylactic uses- (lyophilizedinducer 1 OP (1 ml) intramuscular) Indications Suitable administrationmethods Periods of high infection 2 injections before the stresspressure at an interval of 24 hours Stress Travel, examinations andsimilar stresses before or at the same time as protective inoculationsChemotherapy, irradiation 1 injection each day or every (reduction orprevention of 2nd day until the treatment is secondary reactions)complete or until recovery Operations (improvement in wound healing)Maintenance of optimal 1-2 injections per month at an defenses andhemodynamics interval of 24 hours Prophylaxis of cancer and hepatitidesImprovement in wellbeing

TABLE 2 Clinical results with a monoparamunity inducer from attenuatedmyxoma cell culture virus in humans -therapeutic uses- (lyophilizedinducer 1 OP (1 ml) intramuscular) Indications Suitable administrationmethods Herpetic diseases 1 injection per day for 3-5 (zoster,infectious days or until the symptoms mononucleosis, Herpes simplex,etc.) disappear; then one injection every 2nd or 3rd day until completerecovery Chronic hepatitides a “course” each month: 3 injections atintervals each of 24 hours Influenzal infections 1 administration perday until viral and bacterial mixed the symptoms disappear, then 1infections (in combination with injection every 2nd day untilantibiotics or chemotherapy) complete recovery Immunodeficiencies and 1.intensive treatment for 5-10 days: dysregulation of the defense 1injection per day systems (e.g. during or after 2. then 2 injections perweek chemotherapy) at an interval of 24 hours (treatment over a longerperiod possible) Endotoxin damage 1 injection a day for 7 days or untilrecovery

TABLE 3 Effect of paramunization with myxoma inducer (PIND-MYXO) inpatients with low immune parameters (7 days after PIND-MYXOadministration) Patients' data Parameter Patient Diagnosis/Therapy(normal range) Day 0 Day 7 A.S. Immunosuppression leukocytes 4000 9400female, (4000-10000/μl) 52 years D.B. Ulcerative colitis lymphocytes 6201360 female, cortisone therapy (900-3000/μl) 54 years CD4 cells 400 920(500-1800/μl) CD8 cells 80 210 (100-1000/μl) U.S. Immunologicalleukocytes 3800 7400 male, impairment (4000-10000/μl) 38 years S.C.Metastatic leukocytes 3800 9900 male, prostate carcinoma (4000-10000/μl)43 years radiotherapy B.M. Susceptibility to cytotoxic cells 0 248female, infections (30-360/μl) 56 years

TABLE 4 Affect of paramunization with Myxoma inducer (PIND-MYXO) inpatients with elevated immune parameters (7 days after PIND-MYXOadministration) Parameter Patient Diagnosis/Therapy (normal range) Day 0Day 7 G.P., Cervical, breast leukocytes 12600 8600 female, carcinoma,(4000-10000/μl) 59 years susceptibility to granulocytes 8420 5930infections (2400-6400/μl) C.H. Psychosomatic leukocytes 12700 6000female, syndrome, (4000-10000/μl) 51 years obesity granulocytes 92704760 (2400-6400/μl)

1. A method of preparing a pharmaceutical preparation for use in a humancomprising purifying an attenuated myxomavirus, inactivating themyxomavirus, and lyophilizing the inactivated virus to generate apharmaceutical preparation suitable for use in a human.
 2. The method ofclaim 1, wherein the pharmaceutical preparation is a buccal tablet. 3.The method of claim 2, wherein the tablet comprises Kollidon 25, urea,sorbitol, polyethylene glycol 6000, potassium dihydrogenphosphate,disodium hydrogenphosphate, tyrospirol tablet essence, or magnesiumstearate.
 4. The method of claim 1, wherein the pharmaceuticalpreparation is in dry form.
 5. The method of claim 1, wherein thepharmaceutical preparation is in liquid form.
 6. The method of claim 5,wherein the lyophilized, inactivated virus is resuspended in water. 7.The method of claim 1, wherein the pharmaceutical preparation issuitable for human parenteral injection.
 8. The method of claim 1,wherein the pharmaceutical preparation is a cream for application to thehuman skin.
 9. The method of claim 1, wherein the myxomavirus ispurified by centrifugation.
 10. The method of claim 1, wherein themyxomavirus is inactivated by treatment with beta-propiolactone.
 11. Themethod of claim 10, wherein the beta-propiolactone is at a concentrationof 0.01-1.0%
 12. The method of claim 10, wherein the beta-propiolactoneis at a concentration of 0.05%.
 13. The method of claim 1, wherein themyxomavirus is the attenuated myxomavirus as deposited at ECACC underNo.
 03121801. 14. The method of claim 1, wherein the myxomavirus is amyxomavirus that has lost the receptor properties of one or moremyxomavirus interferon receptor, one or more myxomavirus tumor necrosisfactor receptor, and one or more myxomavirus interleukin receptor. 15.The method of claim 1, wherein the myxomavirus is a myxomavirus that haslost the receptor properties of the myxomavirus cytokine receptorsIFNα-R, IFNγ-R, TNF-R, IL-1-R, IL-2-R, IL-6-R, and IL-12-R.